Project Summary Studies will be carried out with the human replicative helicase, consisting of Cdc45-Mcm2-7-GINS (CMG), and the replicative DNA polymerases in order to catalyze leading and lagging strand synthesis. Our goal is to recapitulate in vitro the in vivo observations suggesting that Pol ? catalyzes leading strand DNA synthesis while Pol ? plus Pol ?-primase complex support lagging strand synthesis. We will utilize a primed 200-nt circle containing only three nucleotides so leading strands can be measured conveniently using dATP incorporation while lagging strand can be followed by dTTP incorporation. We propose that the CMG complex is the protein core at the replication fork. Protein involved in replication, including DNA polymerases, Ctf4, FACT, Tim-Tipin, etc. interact with the CMG complex and constitute the moving replisome. We plan to investigate these interactions and their influence on the CMG helicase activity both in vitro and in vivo. Alterations in components of the CMG complex play important roles in DNA replication. Specific human mutations in Psf1 (a GINS component) have been detected and their effects on the formation of GINS and CMG will be investigated. A number of these mutations lead to developmental defects. We plan to determine whether the level of DNA replication in cells isolated from the Psf1 human mutants contribute to phenotypic defects. 1